Abnormal endothelium dependent vasorelaxation due to reduced nitric oxide bioavailability in the blood vessel wall is a key component of vascular disease associated with hyperlipidemia, hypertension, diabetes mellitus, obesity and atherosclerosis and may be important in the pathogenesis of atherosclerosis. While there are many potential causes of decreased NO bioavailability in the blood vessel wall, decreased NO generation or increased NO degradation via interaction with superoxide anions may play a pivotal role. Gene therapy approaches to atherosclerosis may include systemic delivery of genes to the liver to treat risk factors or local delivery to the vessel wall to enhance NO bioavailability to augment blood flow, enhance new vessel formation or limit cell proliferation in the vessel wall. A gene therapy approach utilizing local delivery of NOS gene to the vessel wall has advantages as nitric oxide has pleiotropic anti-atherogenic effects in the vasculature. In addition, a better understanding of the role of superoxide in endothelial dysftinction in various stages of atherosclerosis may allow the therapeutic effects of superoxide dismutase overexpression to be explored. In this proposal we will test the following hypotheses a) individual NOS isoform may have distinct characteristics for altering vascular reactivity in the normal and diseased blood vessel wall, b) adeno-associated virus vectors can be used to transfer the NOS gene to the vascular wall resulting in long term alteration of vascular reactivity without inflammation and c) long term overexpression of NOS in the blood vessel wall of the hypercholesterolemic rabbit will improve endothelium dependent vasorelaxation and delay the progression of atherosclerosis and d) increased scavenging of superoxide via SOD gene transfer may increase NO bioavailability and reverse lipid-induced endothelial dysfunction. These experiments will determine which NOS isoform is best at altering vascular function, examine the functional effect of prolonged expression of eNOS in the rabbit carotid artery using AAV vectors, examine the effect of NOS overexpression on progression of atherosclerosis and elucidate the role of superoxide in the pathogenesis of endothelial dysfunction in various stages of atherosclerosis.